The present invention relates to cyanovirins and conjugates thereof, matrix-anchored cyanovirins and conjugates thereof, anti-cyanovirin antibodies, compositions comprising same, and methods of using same to remove virus from a sample and to inhibit prophylactically or therapeutically a viral infection of a host.
Acquired immune deficiency syndrome (AIDS) is a fatal disease, reported cases of which have increased dramatically within the past several years. The AIDS virus was first identified in 1983. It has been known by several names and acronyms. It is the third known T-lymphotropic virus (HTLV-III), and it has the capacity to replicate within cells of the immune system, causing profound cell destruction. The AIDS virus is a retrovirus, a virus that uses reverse transcriptase during replication. This particular retrovirus is also known as lymphadenopathy-associated virus (LAV), AIDS-related virus (ARV) and, most recently, as human immunodeficiency virus (HIV). Two distinct families of HIV have been described to date, namely HIV-1 and HIV-2. The acronym HIV is used herein to refer to human immunodeficiency viruses generically.
HIV exerts profound cytopathic effects on the CD4+ helper/inducer T-cells, thereby severely compromising the immune system. HIV infection also results in neurological deterioration and, ultimately, in death of infected individuals. Tens of millions of people are infected with HIV worldwide, and, without effective therapy, most of these are doomed to die. During the long latency, the period of time from initial infection to the appearance of symptoms, or death, due to AIDS, infected individuals spread the infection further, by sexual contacts, exchanges of contaminated needles during i.v. drug abuse, transfusions of blood or blood products, or maternal transfer of HIV to a fetus or newborn. Thus, there is not only an urgent need for effective therapeutic agents to inhibit the progression of HIV disease in individuals already infected, but also for methods of prevention of the spread of HIV infection from infected individuals to noninfected individuals. Indeed, the World Health Organization (WHO) has assigned an urgent international priority to the search for an effective anti-HIV prophylactic virucide to help curb the further expansion of the AIDS pandemic (Balter, Science 266, 1312-1313, 1994; Merson, Science 260, 1266-1268, 1993; Taylor, J. NIH Res. 6, 26-27, 1994; Rosenberg et al., Sex. Transm. Dis. 20, 41-44, 1993; and Rosenberg, Am. J. Public Health 82, 1473-1478, 1992).
The field of viral therapeutics has developed in response to the need for agents effective against retroviruses, especially HIV. There are many ways in which an agent can exhibit anti-retroviral activity (e.g., see DeClercq, Adv. Virus Res. 42, 1-55, 1993; DeClercq, J. Acquir. Immun. Def. Synd. 4, 207-218, 1991; and Mitsuya et al., Science 249, 1533-1544, 1990). Nucleoside derivatives, such as AZT, which inhibit the viral reverse transcriptase, are the only clinically active agents that are currently available commercially for anti-HIV therapy. Although very useful in some patients, the utility of AZT and related compounds is limited by toxicity and insufficient therapeutic indices for fully adequate therapy. Also, given the recent revelations about the true dynamics of HIV infection (Coffin, Science 267, 483-489, 1995; and Cohen, Science 267, 179, 1995), it is now increasingly apparent that agents acting as early as possible in the viral replicative cycle are needed to inhibit infection of newly produced, uninfected immune cells generated in the body in response to the virus-induced killing of infected cells. Also, it is essential to neutralize or inhibit new infectious virus produced by infected cells.
Infection of CD4+ cells by HIV-1 and related primate immunodeficiency viruses begins with interaction of the respective viral envelope glycoproteins (generically termed xe2x80x9cgp120xe2x80x9d) with the cell-surface receptor CD4, followed by fusion and entry (Sattentau, AIDS 2, 101-105, 1988; and Koenig et al., PNAS USA 86, 2443-2447, 1989). Productively infected, virus-producing cells express gp120 at the cell surface; interaction of gp120 of infected cells with CD4 on uninfected cells results in formation of dysfunctional multicellular syncytia and further spread of viral infection (Freed et al., Bull. Inst. Pasteur 88, 73, 1990). Thus, the gp120/CD4 interaction is a particularly attractive target for interruption of HIV infection and cytopathogenesis, either by prevention of initial virus-to-cell binding or by blockage of cell-to-cell fusion (Capon et al., Ann. Rev. Immunol. 9, 649-678, 1991). Virus-free or xe2x80x9csolublexe2x80x9d gp120 shed from virus or from infected cells in vivo is also an important therapeutic target, since it may otherwise contribute to noninfectious immunopathogenic processes throughout the body, including the central nervous system (Capon et al., 1991, supra; and Lipton, Nature 367, 113-114, 1994). Much vaccine research has focused upon gp120; however, progress has been hampered by hypervariability of the gp120-neutralizing determinants, and consequent extreme strain-dependence of viral sensitivity to gp120-directed antibodies (Berzofsky, J. Aca. Immun. Def. Synd. 4, 451-459, 1991). Relatively little drug discovery and development research has focused specifically upon gp120. A notable exception is the considerable effort that has been devoted to truncated, recombinant xe2x80x9cCD4xe2x80x9d proteins (xe2x80x9csoluble CD4xe2x80x9d or xe2x80x9csCD4xe2x80x9d), which bind gp120 and inhibit HIV infectivity in vitro (Capon et al., 1991, supra; Schooley et al., Ann. Int. Med. 112, 247-253, 1990; and Husson et al., J. Pediatr. 121, 627-633, 1992). However, clinical isolates, in contrast to laboratory strains of HIV, have proven highly resistant to neutralization by sCD4 (Orloff et al., AIDS Res. Hum. Retrovir. 11, 335-342, 1995; and Moore et al., J. Virol. 66, 235-243, 1992). Initial clinical trials of sCD4 (Schooley et al., 1990, supra; and Husson et al., 1992, supra), and of sCD4-coupled immunoglobulins (Langner et al., Arch. Virol. 130, 157-170, 1993), and likewise of sCD4-coupled toxins designed to bind and destroy virus-expressing cells (Davey et al., J. Infect. Dis. 170, 1180-1188, 1994; and Ramachandran et al., J. Infect. Dis. 170, 1009-1113, 1994), have been disappointing. Newer gene-therapy approaches to generating sCD4 directly in vivo (Morgan et al., AIDS Res. Hum. Retrovir. 10, 1507-1515, 1994) will likely suffer similar frustrations.
In this regard, it would be advantageous to develop methods and compositions to protect cells against initial infection of a virus. To date, no practical protocols have been developed to produce immunological protection against HIV. The major hurdles associated with the induction of protective immunity against the virus include the inability to separate infectious and non-infectious viral particles for use in a composition that can induce an immune response, such as a vaccine composition. The presence of such non-infectious viral particles in serum samples has been observed and are presumably the result of error-prone replication processes for many viruses. The use of genetically-engineered replication-incompetent virus as a vaccine composition has been proposed to obviate the need to separate infectious and non-infectious viral particles. However, it is unclear how manipulation of the viral genome affects the presentation of antigens on the viral surface. It is entirely possible that the repertoire of viral antigens on a genetically modified replication-incompetent virus would not be characteristic of a naturally occurring virus. It also has been proposed to use incomplete particles to induce an immune response against intact virus. Yet, an immune response to mere pieces of virus cannot be as comprehensive or complete as an immune response to an intact, naturally occurring virus.
Therefore, new classes of antiviral agents, to be used alone or in combination with AZT and/or other available antiviral agents, are needed for effective antiviral therapy against AIDS. New agents, which may be used to prevent HIV infection, are also important for prophylaxis. In both areas of need, the ideal new agent(s) would act as early as possible in the viral life cycle; be as virus-specific as possible (i.e., attack a molecular target specific to the virus but not the host); render the intact virus noninfectious; prevent the death or dysfunction of virus-infected cells; prevent further production of virus from infected cells; prevent spread of virus infection to uninfected cells; be highly potent and active against the broadest possible range of strains and isolates of HIV; be resistant to degradation under physiological and rigorous environmental conditions; and be readily and inexpensively produced on a large-scale basis.
Accordingly, it is an object of the present invention to provide a method of removing virus, in particular infectious virus, such as an immunodeficiency virus, specifically human immunodeficiency virus, e.g., HIV-1 or HIV-2, from a sample.
It is another object of the present invention to provide a composition comprising a naturally-occurring, non-infectious virus, such as a composition obtained in accordance with the above-described method.
It is yet another object to the present invention to provide a method of inducing an immune response to a virus in an animal.
A further object of the present invention is to provide a composition comprising a solid support matrix to which is attached a cyanovirin or a conjugate thereof or an antibody thereto.
A still further object of the present invention is to provide a conjugate comprising a cyanovirin coupled to an anti-cyanovirin antibody or at least one effector component. A related object is to provide a composition comprising such a conjugate.
Another object of the present invention is to provide methods of using such conjugates and matrix-anchored cyanovirins and conjugates thereof in the treatment of viral infection and the induction of an immune response.
These and other objects and advantages of the present invention, as well as additional inventive features, will become apparent from the description provided herein.
The present invention provides, among other things, a method of removing virus from a sample. The method comprises contacting a sample with a composition comprising an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate, any one of which is attached to a solid support matrix. The antiviral protein or antiviral peptide comprises at least nine contiguous amino acids of SEQ ID NO: 2 that bind to the virus. The method further comprises separating the sample and the composition, whereupon the virus is removed from the virus.
The present invention further provides a composition comprising a solid support matrix to which is attached an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate, in which the antiviral protein or antiviral peptide comprises at least nine contiguous amino acids of SEQ ID NO: 2 that bind to a virus. In this regard, the present invention also provides a method of administering such a composition to a host to inhibit prophylactically or therapeutically a viral infection.
A conjugate comprising an isolated and purified antiviral protein or antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2 that bind to a virus, coupled to an anti-cyanovirin antibody or at least one effector component, which can be the same or different, selected from the group consisting of polyethylene glycol, dextran and albumin, and a composition comprising such a conjugate are also provided. Also provided is a method of inhibiting prophylactically or therapeutically a viral infection of a host. The method comprises administering to the host an antiviral effective amount of such a conjugate.
A matrix-anchored anti-cyanovirin antibody is also provided. The antibody binds to an epitope of an antiviral protein or an antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO: 2. The antibody is attached to a solid support matrix. In this regard, the present invention also provides a method of removing virus from a sample. The method comprises contacting the sample with a composition comprising an isolated and purified antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate in which the antiviral peptide or antiviral protein comprises at least nine contiguous amino acids of SEQ ID NO: 2 that bind to a virus. The method further comprises contacting the sample with the matrix-anchored anti-cyanovirin antibody and then separating the matrix-anchored anti-cyanovirin antibody and sample, whereupon the virus is removed from the sample.